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Mechanism of hepatic megamitochondria formation by ammonia derivatives. Correlation between structure of chemicals and their ability to induce the formation of megamitochondria.

Correlation between the chemical structure and the ability to induce hepatic megamitochondria formation was studied by feeding mice and rats diets containing a wide spectrum of ammonia derivatives. Ammonia derivatives with electron-releasing groups, such as hydrazine, phenylhydrazine, hydroxylamine and aniline were effective in inducing megamitochondria. Ammonia derivatives with electron-withdrawing groups, such as formamide, sulfamic acid, acetamide were ineffective in inducing megamitochondria. Inducibility of ammonia derivatives with electron-releasing groups plus electron-withdrawing groups for the megamitochondria formation was dependent upon nucleophilicity of the chemical: 2,4-dinitrophenylhydrazine induced megamitochondria, while acetanilide did not induce megamitochondria. The megamitochondria formation induced by ammonia derivatives was a reversible process. Freeze-fracture studies on megamitochondria indicated that megamitochondria were formed by the fusion of adjacent mitochondria. Phosphorylating capacity of megamitochondria (hydrazine-induced megamitochondria, for example) were normal despite morphological changes. These data might suggest that the nucleophilicity of chemicals plays a key role in the induction of hepatic megamitochondria. These data might also suggest that the phenomenon is an adaptive process to changes of intracellular milieu.

Ammonia↗

Mechanism of the formation of megamitochondria induced by copper-chelating agents. I. On the formation process of megamitochondria in cuprizone-treated mouse liver.

Processes of the formation of cuprizone-induced megamitochondria in mouse liver have been studied in detail by electron microscopy. The earliest change observed was the presence of large intramitochondrial granules. The next stage was the formation of myelin figures by which mitochondria were apparently connected. The third stage was characterized by megamitochondria connected with each other by their outer membranes. Continuity of mitochondria were further examined by serial sections, and megamitochondria were proved to be connected to each other far more frequentlythan expected on one plane of section. A model for the mechanism of megamitochondrial formation is proposed based on electron microscopic evidences, involving the fusion of mitochondrial membranes. Possibility is also discussed that cuprizone-induced megamitochondria may fuse to one single branching mitochondrion.

Adrenal Glands↗

Selenium affects biosilica formation in the demosponge Suberites domuncula. Effect on gene expression and spicule formation.

Selenium is a trace element found in freshwater and the marine environment. We show that it plays a major role in spicule formation in the demosponge Suberites domuncula. If added to primmorphs, an in vitro sponge cell culture system, it stimulates the formation of siliceous spicules. Using differential display of transcripts, we demonstrate that, after a 72-h exposure of primmorphs to selenium, two genes are up-regulated; one codes for selenoprotein M and the other for a novel spicule-associated protein. The deduced protein sequence of selenoprotein M (14 kDa) shows characteristic features of metazoan selenoproteins. The spicule-associated protein (26 kDa) comprises six characteristic repeats of 20 amino acids, composed of 10 distinct hydrophobic regions ( approximately 9 amino acids in length). Recombinant proteins were prepared, and antibodies were raised against these two proteins. Both were found to stain the central axial filament, which comprises the silicatein, as well as the surface of the spicules. In the presence of selenium, only the genes for selenoprotein M and spicule-associated protein are up-regulated, whereas the expression of the silicatein gene remains unchanged. Finally we show that, in the presence of selenium, larger silica aggregates are formed. We conclude that selenium has a stimulatory effect on the formation of siliceous spicules in sponges, and it may be involved in the enzymatic synthesis of biosilica components.

Amino Acid Sequence↗

Isolated star formation: from cloud formation to core collapse.

The formation of stars is one of the most fundamental problems in astrophysics, as it underlies many other questions, on scales from the formation of galaxies to the formation of the solar system. The physical processes involve the turbulent behavior of a partially ionized medium containing a non-uniform magnetic field. Current debate centers around the time taken for turbulence to decay and the relative importance of the roles played by magnetic fields and turbulence. Technological advances such as millimeter-wave cameras have made possible observations of the temperature and density profiles, and statistical calculations of the lifetimes, of objects collapsing under their own self-gravity and those on the verge of collapse. Increased computing power allows more complex models to be made that include magnetic and turbulent effects. No current model can reproduce all of the observations.

Journal Article↗

Pseudoglandular formations in clot sections from fine needle aspirates--an artefact caused by bubble formation during aspiration.

AIMS: To report the occurrence of an uncommon artefact producing pseudoglandular formations in clot sections from haemorrhagic fine needle aspirations. METHODS: All available histological material from 610 fine needle aspirations by pathologists (23 g needle) over a five year period was reviewed. The frequency and associations of the pseudoglandular artefact was assessed. RESULTS: Clot sections were prepared in 41 of the 610 cases (7%). Bubbles were present in the clots in 22 of these cases (54%), and in three cases (7%) these were lined by lymphocytes creating pseudoglandular formations. These were two lymph node aspirates and one thyroid aspirate. In four further cases lesser numbers of cells partly lined some of the bubbles; these were lymphocytes, macrophages, or in one case, thyroid epithelial cells. CONCLUSIONS: When clot sections are prepared in cases of haemorrhagic fine needle aspiration, bubbles are often produced during suction; these can on occasion become lined by lymphocytes or other cells, leading to pseudoglandular formations. Recognition of this artefact will prevent unnecessary further investigation of their nature.

Adult↗

Impairment of T lymphocyte colony formation in Hodgkin's disease: effect of soluble inhibitory factors on normal T lymphocyte colony formation potential.

PHA-induced T lymphocyte colonies from peripheral blood mononuclear cells of untreated Hodgkin's disease (HD) patients and normal healthy donors were assayed by one-step stimulation in microagar capillary cultures. A significant depression in the T cell colony formation was observed in HD patients in comparison with normal healthy donors. Clinical staging of the disease had no influence on this abnormality. Preincubation of HD lymphocytes for 24 h in tissue culture medium did not produce any appreciable recovery in the colony formation potential. However, in the presence of 24-hour culture supernatants of HD mononuclear cells, there was significant inhibition in the colony formation by lymphocytes obtained from normal healthy donors. Significance of these observations is discussed.

Hodgkin Disease↗

Local monocyte chemoattractant protein-1 therapy increases collateral artery formation in apolipoprotein E-deficient mice but induces systemic monocytic CD11b expression, neointimal formation, and plaque progression.

Monocyte chemoattractant protein-1 (MCP-1) stimulates the formation of a collateral circulation on arterial occlusion. The present study served to determine whether these proarteriogenic properties of MCP-1 are preserved in hyperlipidemic apolipoprotein E-deficient (apoE-/-) mice and whether it affects the systemic development of atherosclerosis. A total of 78 apoE-/- mice were treated with local infusion of low-dose MCP-1 (1 microg/kg per week), high-dose MCP-1 (10 microg/kg per week), or PBS as a control after unilateral ligation of the femoral artery. Collateral hindlimb flow, measured with fluorescent microspheres, significantly increased on a 1-week high-dose MCP-1 treatment (PBS 22.6+/-7.2%, MCP-1 31.3+/-10.3%; P<0.05). These effects were still present 2 months after the treatment (PBS 44.3+/-4.6%, MCP-1 56.5+/-10.4%; P<0.001). The increase in collateral flow was accompanied by an increase in the number of perivascular monocytes/macrophages on MCP-1 treatment. However, systemic CD11b expression by monocytes also increased, as did monocyte adhesion at the aortic endothelium and neointimal formation (intima/media ratio, 0.097+/-0.011 [PBS] versus 0.257+/-0.022 [MCP-1]; P<0.0001). Moreover, Sudan IV staining revealed an increase in aortic atherosclerotic plaque surface (24.3+/-5.2% [PBS] versus 38.2+/-9.5% [MCP-1]; P<0.01). Finally, a significant decrease in the percentage of smooth muscle cells was found in plaques (15.0+/-5.2% [PBS] versus 5.8+/-2.3% [MCP-1]; P<0.001). In conclusion, local infusion of MCP-1 significantly increases collateral flow on femoral artery ligation in apoE-/- mice up to 2 months after the treatment. However, the local treatment did not preclude systemic effects on atherogenesis, leading to increased atherosclerotic plaque formation and changes in cellular content of plaques.

Animals↗

Apatite formation on/in hydrogel matrices using an alternate soaking process: II. Effect of swelling ratios of poly(vinyl alcohol) hydrogel matrices on apatite formation.

In our previous study, we reported a novel method of apatite formation on/in a three-dimensional hydrogel matrix. Using this method, bone-like apatite could be formed on/in the hydrogel matrix under normal conditions in vitro. A poly(vinyl alcohol) (PVA) gel was used as a model matrix. The method consists of two steps: first, water is transformed in a PVA gel with a CaCl2/Tris-HCl aqueous solution (pH 7.4) and second, the gel is soaked in a Na2HPO4 aqueous solution. In the present study, we report a detailed study of the effects of the swelling ratios of PVA gels on apatite formation. Cross-sectional observations and gravimetric measurements of PVA gels with various swelling ratios were done. The amount of apatite formed on/in PVA gels increased almost linearly with an increase in the reaction cycles. The rates of apatite formation on/in PVA gels largely depended on the swelling ratios, which were approximately 0.48, 0.61, 1.28, and 1.55 mg per cycle for swelling ratios of 4.1, 10.4, 16.8, and 30.1, respectively. The apatite content in PVA-apatite composites that was obtained by this method also increased with an increase of the reaction cycles. After six reaction cycles, a PVA gel with a high swelling ratio contains approximately 70 wt% of formed apatite in the composite. On the other hand, a gel with a low swelling ratio contains about 15 wt% of formed apatite in the composite. Cross-sectional views of the PVA gels after each cycle showed that apatite crystals were formed, not only on the surface of the gel but also within it after fifteen reaction cycles. The hydrogel-apatite composites that were obtained using an alternative soaking process will be useful as not only bone substitute materials but also as soft tissue adhesive materials.

Apatites↗

Aberrant fibrin formation and cross-linking of fibrinogen Nieuwegein, a variant with a shortened Aalpha-chain, alters endothelial capillary tube formation.

A congenital dysfibrinogenemia, fibrinogen(Nieuwegein), was discovered in a young man without any thromboembolic complications or bleeding. A homozygous insertion of a single nucleotide (C) in codon Aalpha 453 (Pro) introduced a stop codon at position 454, which resulted in the deletion of the carboxyl-terminal segment Aalpha 454-610. The ensuing unpaired cysteine at Aalpha 442 generated fibrinogen-albumin complexes of different molecular weights. The molecular abnormalities of fibrinogen(Nieuwegein) led to a delayed clotting and a fibrin network with a low turbidity. Electron microscopy confirmed that thin fibrin bundles were organized in a fine network. The use of fibrinogen(Nieuwegein)-derived fibrin (fibrin(Nieuwegein)) in an in vitro angiogenesis model resulted in a strong reduction of tube formation. The ingrowth of human microvascular endothelial cells (hMVEC) was independent of alpha(v)beta(3), indicating that the reduced ingrowth is not due to the absence of the RGD-adhesion site at position Aalpha 572-574. Rather, the altered structure of fibrin(Nieuwegein) is the cause, since partial normalization of the fibrin network by lowering the pH during polymerization resulted in an increased tube formation. Whereas factor XIIIa further decreased the ingrowth of hMVEC in fibrin(Nieuwegein), tissue transglutaminase (TG), which is released in areas of vessel injury, did not. This is in line with the absence of the cross-linking site for TG in the alpha-chains of fibrinogen(Nieuwegein). In conclusion, this newly discovered congenital dysfibrinogenemia has a delayed clotting time and leads to the formation of an altered fibrin structure, which could not be cross-linked by TG and which is less supportive for ingrowth of endothelial cells.

Adult↗

Catechol estrogen formation by brain tissue: characterization of a direct product isolation assay for estrogen-2- and 4-hydroxylase activity and its application to studies of 2- and 4-hydroxyestradiol formation by rabbit hypothalamus.

A direct product isolation assay for quantifying the formation of 2- and 4-hydroxyestradiol (2-OHE2 and 4-OHE2) from [6,7-3H]estradiol by rabbit hypothalami in vitro was developed, and the assay was used to characterize some properties of estrogen-2- and 4-hydroxylase activity in this tissue. The reaction was carried out under conditions that minimized further metabolism of enzymatically formed catechol estrogens. A simple two-step separation procedure, involving the use of a neutral alumina column, followed by thin layer chromatography, was developed to isolate the enzymatically formed catechol estrogens in a radiochemically homogeneous form. The detergent, Tween-80, was found to activate the enzyme and was used routinely at a concentration of 0.1% in the assay. The formation of 2-OHE2 was linear up to 10 min and with increasing protein concentrations up to 150 micrograms/incubation. Similar values were obtained for 4-OHE2. Maximum velocities (Vmax) for the formation of 2- and 4-OHE2 were 190 and 270 pmol/mg protein . 10 min, respectively. The apparent Km values with respect to estradiol for 2-OHE2 and 4-OHE2 were 125 and 150 microM, respectively. The highest specific activity for the enzyme was present in the 100,000 X g supernatant (S3), while the activity in the microsomal fraction (P3) was less than that in the original homogenate. Enzyme activity depended on the presence of NADPH and oxygen and was inhibited by CO as well as by high concentrations of SKF-525A. Estrogen-2- and 4-hydroxylase activity in rabbit hypothalamus differed from that in rat liver in two respects. In the liver, enzyme activity was localized in the microsomal fraction and was virtually abolished by Tween-80. In contrast, enzyme activity in rabbit hypothalamus was maximal in the soluble fraction (100,000 X g supernatant)and was stimulated by the detergent.

Animals↗

Formation and persistence of 8-oxoguanine in rat lung cells as an important determinant for tumor formation following particle exposure.

Exposure of rats to quartz (or various other particles) can lead to the development of lung tumors. At the moment, the mechanisms involved in particle-induced tumor formation are not clarified. However, it is suggested that inflammation, in conjunction with the production of reactive oxygen species (ROS) and an enhancement of epithelial cell proliferation, may play a key role in the development of lung tumors. ROS induces 8-oxoguanine (8-oxoGua) and other mutagenic DNA oxidation products, which can be converted to mutations in proliferating cells. Mutation formation in cancer-related genes is a critical event with respect to tumor formation. In this study we investigated the effects of quartz (DQ12) and of the nontumorigenic dust corundum on the induction of 8-oxoGua in the DNA of rat lung cells, as well as on cell proliferation and pulmonary inflammation. Wistar rats were exposed by intratracheal instillation to quartz (2.5 mg/rat) or corundum (2.5 mg/rat) suspended in physiological saline; control animals exposed to physiological saline or left untreated. Measurements were carried out 7, 21, and 90 days after the exposures. 8-oxoGua levels were determined in lung tissue sections at the single cell level by immunocytological assay using a rabbit anti-8-oxoGua antibody. After exposure to quartz, 8-oxoGua levels were significantly increased at all time points of investigation. Additionally, we observed inflammation and an enhanced cell proliferation. Exposure to corundum had no adverse effects on the lung; neither increased 8-oxoGua levels nor enhanced cell proliferation or inflammation were detected. These observations support the suggestion that inflammation associated with increased 8-oxoGua levels in lung cells and increased cell proliferation is an important determinant for particle-induced development of lung tumors in the rat.

8-Hydroxy-2'-Deoxyguanosine↗

Extra-anatomical bypass formation and exclusion of thoracic aortic aneurysms by "paired clamp method": the development of hind leg paralysis, the thrombus formation and the pressure ratio between the excluded cavity and the aorta.

The extra-anatomical bypass formation and the exclusion of thoracic aortic aneurysms by the "paired clamp method" applied to the thoracic aorta of mongrel dogs, and the development of hind leg paralysis was studied experimentally in relation to the ratio between the mean excluded cavity pressure and the mean aortic pressure ratio (EA-ratio). The relationship between thrombus formation in the excluded cavity and the EA-ratio was also studied. Animals were divided into 4 groups: Group 1 underwent the cross-clamping of the descending thoracic aorta for 8 min; Group 2 underwent exclusion of the entire thoracic aorta under permanent bypass; Group 3 underwent ligation of 2/3 of the proximal intercostal arteries which branched from the excluded thoracic aorta; and Group 4 underwent division of the excluded thoracic aorta into 3 parts by ligation. No animals in group 1 developed paralysis. When EA-ratios were higher than 0.48, animals in groups 2 and 3 were not paralyzed, whereas all but 1 animals with EA-ratios lower than or equal to 0.48 were paralyzed. There was statistically significant difference between the EA-ratio of the non-paralyzed animals and that of the paralyzed animals in groups 2 and 3. Four out of 5 animals in group 4 were paralyzed. However, there was no statistically significant difference between the EA-ratio in these 4 paralyzed animals and that in the non-paralyzed animals in groups 2 and 3. When the EA-ratio was lower than or equal to 0.59, all but 1 excluded cavities of groups 2 and 3 animals were fully thrombosed 7 or 8 days after the operation. None of these animals showed the aggravation of the paralysis during the observation period and, conversely, the paralysis of almost all animals was ameliorated. These results suggested that the EA-ratio is useful in predicting the development of paralysis and thrombus formation in the excluded cavity after the operation of thoracic aortic aneurysms by the "paired clamp method".

Animals↗

Substrate specificity for formation of cholesterol ester conjugates from fenvalerate analogues and for granuloma formation.

1. The substrate specificity of microsomal carboxyesterase(s) responsible for the formation of cholesteryl [2R]-2-(4-chlorophenyl) isovalerate from fenvalerate was investigated by incubating mouse kidney microsomes with 14C-cholesterol and the following substrates: fenvalerate isomers, fenvalerate analogues, other pyrethroids, methoprene and cycloprate analogues. Among the four isomers of fenvalerate, only the [2R, alpha S]-isomer yielded a cholesterol ester, being identical with the result obtained in the in vivo study. Some fenvalerate analogues produced cholesterol ester conjugates, but no other pyrethroids nor methoprene produced such conjugates. Some cycloprate analogues gave the corresponding cholesterol ester, the yields of which were dependent on their carbon-chain lengths. 2. Cholesterol ester formation in vitro from these fenvalerate analogues was well correlated with granuloma formation observed when the analogues were given to mice at 3000 ppm for a month. 3. Steroids other than cholesterol were also investigated as acceptors of the acid moiety of the [2R, alpha S]-isomer by incubating solubilized carboxyesterase(s) with the [2R, alpha S]-isomer in the presence of egg lecithin and several steroids. Dehydroisoandrosterone and pregnenolone were found to give the corresponding ester conjugates.

Animals↗

Chemoprevention of colon cancer carcinogenesis by balsalazide: inhibition of azoxymethane-induced aberrant crypt formation in the rat colon and intestinal tumor formation in the B6-Min/+ mouse.

Non-steroidal anti-inflammatory drugs (NSAIDs) including aspirin have been shown to suppress colon carcinogenesis and in some cases reduce the size of colorectal polyps. Balsalazide disodium (BSZ) is a colon-specific prodrug of the salicylate, 5-aminosalicylic acid. The aim of the present study was to test the chemopreventive activity of BSZ in two established animal models of colon tumorigenesis, azoxymethane-induced aberrant crypt formation in the rat and intestinal tumor formation in the B6-Min/+ mouse. Aberrant crypt foci (ACF) were induced in Fischer 344 rats via 2 subcutaneous injections of azoxymethane (20 mg/kg). BSZ was supplied in the drinking water for 8 weeks and ACF quantitated. B6-Min/+ mice were treated from 55 days of age for 90 days and intestinal tumors scored for number, size and location. BSZ treatment of AOM-injected rats reduced ACF formation in a dose-dependent manner by 60% with the greatest effect observed on ACF with 4 or more crypts. In B6-Min/+ mice a dose-dependent reduction of intestinal tumor number was observed which reached 80% in the distal small intestine and colon. A preliminary mechanistic study in cultured human colon cancer cells showed that both BSZ and 5-ASA inhibited colon cancer cell proliferation in vitro. However, 5-ASA but not BSZ produced changes consistent with the induction of apoptosis. BSZ produces a dose-dependent chemopreventive effect on colon carcinogenesis. A possible mechanism is consistent with the inhibition of cellular proliferation and the induction of apoptosis.

Aminosalicylic Acids↗

[Study of bone formation in fetal rat parietal bone using serum-free bone-forming organ culture system: effect of insulin on bone formation].

Serum-free bone-forming organ culture system using twenty-day fetal rat parietal bones was established. The effects of insulin on bone formation were studied in this system. Bones were cultured on grids and placed on a rocking platform under an aerobic condition. This condition increased the bone-forming activities of cultured rat parietal bones. The calcium content, dry weight of bone, and lactate produced by bone tissue in the medium were measured to study the precise bone-forming process of vital bones. Using this improved bone-forming organ culture system, insulin increased calcium content and dry weight of bone. These effects were maximum in bones treated with insulin at 10(-6) M. Histo-morphometrical analysis showed that the areas of mineralized bone and bone matrix increased, and that many osteoblasts and few osteoclasts appeared in bones treated with insulin at 10(-6) M or higher. Insulin inhibited the release of 45Ca into the medium. From these results, it was suggested that insulin has a stimulatory effect on bone formation, enhancing both bone mineralization and bone matrix formation.

Animals↗

Fibrous cap formation or destruction--the critical importance of vascular smooth muscle cell proliferation, migration and matrix formation.

Endothelial activation and infiltration of monocyte macrophages are essential prerequisites for fibrous cap formation, which comprises proliferation and migration of smooth muscle cells and net matrix deposition. Macrophage foam cells and endothelium act as a source of growth factors and chemoattractants for smooth muscle cells. However, growth factors alone do not stimulate smooth muscle cell proliferation or migration. This requires, in addition, the remodelling of the extracellular matrix, at least partly mediated by metalloproteinases. In particular, loss of basement membrane components and contact with the interstitial matrix appears to be required to release a brake on proliferation and migration exerted by the basement membrane. Unless there is a change in the phenotype of macrophages in advanced lesions, it is not clear why fibrous cap destruction rather than formation should take place in macrophage-rich shoulder regions of plaques. Impaired cap formation caused by smooth muscle senescence, mummification and propensity to apoptosis may be as important as increased cap destruction in promoting plaque rupture.

Animals↗

Proteolytic formation of either of the two prothrombin activation intermediates results in formation of a hirugen-binding site.

Hirugen, a synthetic dodecapeptide corresponding to the carboxyl-terminal amino acids 53-64 of hirudin, binds within a deep groove in thrombin that contains a cationic region referred to as the anion-binding exosite. This region is important in many of the binary interactions of thrombin with macromolecular substrates and cofactors. Fluorescein-labeled hirugen was used to probe which steps in the prothrombin activation process generate this anion-binding exosite. Two activation cleavage sites exist in bovine prothrombin. Cleavage at Arg274-Thr275 releases the activation fragments to generate the thrombin precursor, prethrombin 2. Cleavage of prothrombin within a disulfide loop at Arg323-Ile324 leads to formation of meizothrombin with no loss of peptide material but with formation of amidolytic activity. Cleavage of the same bond in prethrombin 2 generates thrombin. Hirugen, labeled at the amino terminus with fluorescein isothiocyanate, does not bind to prothrombin but does bind to thrombin (Kd = 9.6 +/- 1.2 x 10(-8) M), prethrombin 2 (Kd = 1.3 +/- 0.1 x 10(-7) M), thrombin-fragment-2 complex (Kd = 1.1 +/- 0.2 x 10(-6) M), and meizothrombin (Kd = 1.6 +/- 0.5 x 10(-8) M). Prothrombin fragment-2 and hirugen both bind independently to thrombin. A ternary complex can form with hirugen and fragment-2 and either thrombin or prethrombin 2, suggesting that fragment-2 and hirugen bind to discrete sites. Hirugen also alters the active site conformation of thrombin as detected by modulation of synthetic substrate hydrolytic activity. These studies suggest that conformational changes, rather than alleviating steric hindrance, are responsible for the formation of the hirugen-binding site during prothrombin activation. Furthermore, this conformational change can be effected by the cleavage of either of the two bonds required for activation of prothrombin.

Amino Acid Sequence↗

Meizothrombin formation during factor Xa-catalyzed prothrombin activation. Formation in a purified system and in plasma.

Meizothrombin and thrombin formation were quantitated during factor Xa-catalyzed activation of human prothrombin in reaction systems containing purified proteins and in plasma. In the purified system considerable amounts of meizothrombin accumulated when prothrombin was activated by factor Xa (with or without accessory components) under initial steady state conditions. The ratio of the rates of meizothrombin and thrombin formation was not influenced by variation of the pH, temperature, or ionic strength of the reaction medium. When 2 microM prothrombin was activated by the complete prothrombinase complex (factor Xa, factor Va, Ca2+, and phospholipid) 80-90% of the initially formed reaction product was meizothrombin. Lowering the prothrombin concentration from 2 to 0.03 microM caused a gradual decrease in the ratio of meizothrombin/thrombin formation from 5 to 0.6. When the phosphatidylserine content of the phospholipid vesicles was varied between 20 and 1 mol % and prothrombin activation was analyzed at 2 microM prothrombin the relative amount of meizothrombin formed decreased from 85 to 55%. With platelets, cephalin, or thromboplastin as procoagulant lipid, thrombin was the major reaction product and only 30-40% of the activation product was meizothrombin. We also analyzed complete time courses of prothrombin activation both with purified proteins and in plasma. In reaction systems with purified proteins substantial amounts of meizothrombin accumulated under a wide variety of experimental conditions. However, little or no meizothrombin was detected in plasma in which coagulation was initiated via the extrinsic pathway with thromboplastin or via the intrinsic pathway with kaolin plus phospholipid (cephalin, platelets, or phosphatidylserine-containing vesicles). Thus, thrombin was the only active prothrombin activation product that accumulated during ex vivo coagulation experiments in plasma.

Enzyme Activation↗